1. Field of the Invention
The present invention relates to transgenic mice for studying developmental biology and human disease. More specifically, the present invention relates to transgenic mice having a disrupted endogenous endoglin gene.
2. Technical Background
The use and study of laboratory animals has proven useful in understanding human disease. Animals can be kept in controlled conditions to observe disease pathology and to test new treatments for disease. However, animal models have limitations. Chief among these limitations is the large number of genetic differences between animals and humans. These differences sometimes reduce the usefulness of animal models in understanding and developing treatments for human disease. For example, some human diseases are caused by genetic factors that may not naturally occur in laboratory animals. For these and other reasons, transgenic animals have been developed.
A transgenic animal carries a genetic sequence (called a xe2x80x9ctransgenexe2x80x9d or xe2x80x9cheterologous sequencexe2x80x9d) that is not endogenous to that animal. The genetic sequence has been introduced into the germline of the animal or an ancestor of the animal at an early developmental stage.
For example, transgenic mice have been developed with an activated oncogene sequence. These mice are useful models in the study of cancer. Other transgenic animals have been developed which contain and express various genes for human proteins. Yet other transgenic animals have been made in which one or more genes have been mutated by either random or targeted insertion of a transgene or transgenes. These animals may serve as useful models for human diseases that are caused by genetic factors.
Even with a number of transgenic animals being developed, there is a need for animal models for a variety of human diseases. For example, the loss-of-function mutations in the human endoglin gene, ENG, cause hereditary hemorrhagic telangiectasia (HHT1), a disease characterized by vascular malformations. HHT is an autosomal dominant vascular dysplasia characterized by recurrent epistaxis, telangiectasia, gastrointestinal hemorrhage, and pulmonary, cerebral and hepatic arteriovenous malformations. McAllister et al., Nature Genet. 8:345 (1994); Guttmacher et al., N. Engl. J. Med. 33:918 (1995). Endoglin is a transforming growth factor beta (TGF-xcex2) binding protein expressed on the surface of endothelial cells. ENG, the gene responsible for HHT1, encodes an endothelial transmembrane protein that binds to members of the TGF-xcex2 superfamily and their receptor complexes. St.-Jacques et al., Endocrinology 134:2645 (1994); J. R. Westphal et al., J. Invest. Dermatol. 100:27 (1993); H. Yamashita et al., J. Biol. Chem. 269:1995 (1994); Cheifetz et al., J. Biol. Chem. 267:19027 (1992); Barbara et al., J. Biol. Chem. 274:584 (1999). TGF-xcex2 signaling is required for the first stage of vascular development, vasculogenesis, in which the primary capillary network, comprised of interconnected and homogeneously sized endothelial tubes, is formed. Dickson et al., Development 121: 1845 (1995); Oshima, et al., Dev. Biol. 179: 297 (1996). The second stage of vascular development, angiogenesis, involves the remodeling of the primary endothelial network into a mature circulatory system. Folkman and D""Amore, Cell 87:1153 (1996); Yancopoulos et al., Cell 93:661 (1998); Flamme and Risau, Development 116:435 (1992); Pepper, Cytokines and Growth Factor Rev. 8:21 (1993); Kinglsey, Genes Dev. 8:133 (1994); Reddi, Cytokine Growth Factor Rev 8:11 (1997); Massague et al., Trends Cell Biol. 4:172 (1994).
The pathology of HHT1 is little understood. Moreover, since HHT1 results from a malformation of the vascular system, a model for HHT1 may result in greater understanding of other vascular related diseases and angiogenesis in tumors. Therefore, it would be an advancement in the art to provide an animal model for understanding the role of endoglin in vascular development. It would be a further advancement to provide cells from such animals for use in laboratory research.
Such animals and cells are disclosed herein.
The invention provides transgenic mice in which the endogenous endoglin gene is disrupted. In certain embodiments, a transgenic mouse of the present invention is homozygous for endoglin gene disruption. A mouse which is homozygous for the endoglin gene disruption is designated xe2x80x9cEngxe2x88x92/xe2x88x92.xe2x80x9d As a result of the homozygous gene disruption, the mice are unable to produce endoglin, which is a transforming growth factor beta (TGF-xcex2) binding protein expressed on the surface of endothelial cells. Due to this deficiency in endoglin, the development of the vascular system of the mice is arrested. Because of the arrested vascular system development, the Engxe2x88x92/xe2x88x92 mice do not live beyond embryonic (E) day 11.5. These mice are useful for understanding the development of the mammalian vascular system and suggest a pathogenic mechanism for hereditary hemorrhagic telangiectasia. These mice and cells derived from these mice may also be useful for the study of other angiogenesis-related phenomena such as obstructive vascular disease and tumor angiogenesis. These other phenomena are of considerable importance to public health.
A transgenic mouse of the present invention can be created by disrupting the endogenous endoglin gene (ENG). A targeting vector my be designed to replace the first two exons or other portion of ENG with a selectable marker gene sequence or other heterologous sequence by homologous recombination. A selectable marker gene sequence may comprise a gene conferring antibiotic resistance such as neomycin resistance. Other selectable markers are known in the art. In certain embodiments, a heterologous sequence other than a selectable marker is used to disrupt the ENG gene. Such sequences are introduced to destroy or alter the function of the ENG gene and include, for example, stop codons, portions of the ENG gene sequence that have been altered by mutations (including frameshift mutations, insertions, deletions, and point mutations), or sequences that code for portions of heterologous proteins. Insertions that disrupt the ENG gene by homologous recombination but do not employ a selectable marker may nevertheless be detected by screening methods; for example, polymerase chain reaction (PCR) based strategies may be used to screen large numbers of transfected embryonic stem (ES) cells for the desired insertion.
Targeted ES cells are identified and used to generate chimeric mice by morula aggregation. Well-known techniques, such as Southern analysis or PCR, may be used to confirm germline transmission of the targeted allele.
The present invention also provides cells derived from transgenic Engxe2x88x92/xe2x88x92 mice and mouse embryos. A variety of methods for isolating and culturing such cells are known to those of skill in the art. In certain embodiments, a cell is derived from a transgenic mouse embryo homozygous for endogenous endoglin disruption, said disruption resulting from insertion of a selectable marker gene sequence or other heterologous sequence into the genome by homologous recombination, wherein the disruption results in a lack of expression of the endoglin gene product, and wherein the lack of expression of the endoglin gene product results in arrested development of the vascular system of the mouse embryo. In other embodiments of the present invention, the cell derived from a transgenic Engxe2x88x92/xe2x88x92 mouse or embryo is an embryonic stem cell.
A transgenic Engxe2x88x92/xe2x88x92 mouse is deficient in development of the vascular system and does not live past E11.5. Therefore, it may prove advantageous to provide a transgenic mouse hemizygous for disruption of an endogenous endoglin gene, designated as Eng+/xe2x88x92. A transgenic Eng+/xe2x88x92 mouse may be created by the disruption of one copy of the endogenous endoglin gene as discussed above. The disruption may result from the insertion of a selectable marker gene sequence or other heterologous sequence into the genome by homologous recombination. A selectable maker make include a gene sequence conferring resistence to an antibiotic such as neomycin.
The present invention also provides cells derived from an Eng+/xe2x88x92 mouse or embryo and mouse cells that are hemizygous for disruption of the endogenous endoglin gene. In other embodiments of the present invention, the cell derived from a transgenic Eng+/xe2x88x92 mouse is an embryonic stem cell.
These and other features and advantages of the present invention will become more fully apparent from the Detailed Description.